Kv7 channel opener retigabine reduces self‐administration of cocaine but not sucrose in rats

Abstract The increasing rates of drug misuse highlight the urgency of identifying improved therapeutics for treatment. Most drug‐seeking behaviours that can be modelled in rodents utilize the repeated intravenous self‐administration (SA) of drugs. Recent studies examining the mesolimbic pathway suggest that Kv7/KCNQ channels may contribute to the transition from recreational to chronic drug use. However, to date, all such studies used noncontingent, experimenter‐delivered drug model systems, and the extent to which this effect generalizes to rats trained to self‐administer drugs is not known. Here, we tested the ability of retigabine (ezogabine), a Kv7 channel opener, to regulate instrumental behaviour in male Sprague Dawley rats. We first validated the ability of retigabine to target experimenter‐delivered cocaine in a conditioned place preference (CPP) assay and found that retigabine reduced the acquisition of place preference. Next, we trained rats for cocaine‐SA under a fixed‐ratio or progressive‐ratio reinforcement schedule and found that retigabine pretreatment attenuated the SA of low to moderate doses of cocaine. This was not observed in parallel experiments, with rats self‐administering sucrose, a natural reward. Compared with sucrose‐SA, cocaine‐SA was associated with reductions in the expression of the Kv7.5 subunit in the nucleus accumbens, without alterations in Kv7.2 and Kv7.3. Therefore, these studies reveal a reward‐specific reduction in SA behaviour and support the notion that Kv7 is a potential therapeutic target for human psychiatric diseases with dysfunctional reward circuitry.


| INTRODUCTION
Repeated exposure to addictive drugs is known to hyperactivate the mesocorticolimbic ventral tegmental area (VTA), nucleus accumbens (NAc) and prefrontal cortex (PFC) brain regions important for the integration of reinforcing stimuli with goal-directed behaviour. 1,24][5][6][7][8] In neurons, the opening of K v 7 channels triggers a repolarizing M-current associated with spike frequency adaptation. 9,10Retigabine, a K v 7 channel opener, stabilizes M-currents and by positive modulation of the membrane potential restricts the excitability of cortical and mesencephalic dopamine (DA) neurons. 9,10[13] Furthermore, in vitro and in vivo studies show that retigabine can reduce the release of DA from the terminals of mesencephalic dopaminergic neurons 14 and reduce the locomotor activity induced by psychostimulants. 15,16These actions of retigabine are reversed by the K v 7 channel blocker XE-991. 15,17However, administration of retigabine alone does not alter exploratory motility and rotarod behaviour, 18,19 and doses at which retigabine induces anxiolysis can be dissociated from effects on sedation or memory impairment. 18To date, most studies using retigabine to modulate drug-induced changes in DA neurons have used noncontingent methods. 16,202][23][24][25][26][27] However, it remains unknown whether retigabine alters ongoing drug self-administration (SA) behaviour, from which these relapse-like neuroadaptations later emerge.
In this study, we propose that K v 7 channels act as inhibitory regulators of motivated drug behaviour, thus decreasing the reinforcing actions of cocaine during the maintenance of chronic voluntary SA.We first tested this hypothesis in male Sprague Dawley rats to identify a retigabine dose sufficient to reduce the conditioned preference for cocaine and then determined whether lower, similar or higher doses of retigabine reversibly reduced the operant responding for various doses of cocaine.We showed that retigabine reversibly weakens the motivation for cocaine-SA at low to moderate doses on a fixedratio or progressive-ratio (PR) reinforcement schedule.We examined the expression of various K v 7 channel subunits in relevant mesolimbic regions and provided evidence that relative to sucrose-SA, chronic cocaine-SA was associated with a notable reduction in the expression of the K v 7.5 channel subunit in the accumbens.This study shows that the motivation to self-administer cocaine is reduced by a potent activator of K v 7 channels and that the ability to ameliorate this drug behaviour does not necessarily generalize to other nondrug (natural) rewards.

| Subjects
Male Sprague Dawley rats (n = 120; Envigo) weighing 250-275 g were housed individually in a reverse 12-h light/dark cycle (light off at 06:00 AM).All procedures performed were preapproved by the Institutional Animal Care and Use Committee of the University of Arizona and according to the Animal Care Guidelines of the National Institutes of Health for the Care and Use of Laboratory Animals.See Data S1.

| Drugs
Retigabine (Axon Medchem) was dissolved in saline with 10% Tween-80 (Sigma-Aldrich) at concentrations of 2, 5 or 7 mg/mL.Retigabine or vehicle pretreatments were administered 15 min before CPP or SA. 19Doses of retigabine were selected on the basis of previous studies showing similar and higher doses produced no significant decrements in a rotarod test and zero maze exploration. 18,19As the retigabine solution has a purple colour that is clearly discernable from the vehicle solution, we considered the experimenters to be unblinded.Cocaine hydrochloride (Drug Supply Program of the National Institute on Drug Abuse) was prepared in sterile saline.

| Conditioned place preference (CPP)
Using our established procedures, 28 we performed cocaine CPP in a three-chambered system (San Diego Instruments).Using a biased design, rats were assigned to the least preferred chamber for pairing with cocaine (10 mg/kg, i.p.).Rats were assigned randomly to receive either retigabine or vehicle.Conditioning sessions (30 min) occurred twice a day, with one chamber in the morning (Session 1) and the opposite chamber in the afternoon (Session 2).Daily saline and cocaine sessions were counterbalanced with respect to session (Figure 1A).Pretreatments with retigabine (5 mg/kg, i.p.) or vehicle (10% tween-80 in saline) occurred 15 min before each cocaine conditioning session.After the five conditioning days, the rats underwent a 15-min post-test.CPP score was calculated as the difference in the time spent in individual chambers during pre-test and post-test. 29Locomotor activity during the pre-test and post-test was not evaluated.

| Intravenous catheter surgery
Using our published procedures, 9 rats anaesthetised with isoflurane gas (5% for induction; 2.1%-2.5% for maintenance) received a 22 gauge silastic catheter placed in the right jugular vein.See Data S1.
Following the acquisition of cocaine-SA, animals were divided into three groups (1A, 1B and 1C) (Figure 2A).Before retigabine testing, animals in Groups 1A and 1B continued training on an FR1 (2 h) schedule, at a dose of 0.5 mg/kg/infusion (Group 1A) or a lower dose of 0.1 mg/kg/infusion (Group 1B).Animals were habituated to the (i.p.) pretreatment injections with a minimum of 2 days of saline treatments (1 mL/kg, i.p.) before retigabine and every day thereafter if not receiving retigabine (Figure 3A).The half-life of retigabine in rodents is about 3 h, 30 and we allowed a minimum of 2 days between the retigabine tests.
In addition to the 12-session FR1 training (above), animals in Group 1C (Figure 2A) underwent two additional sessions of FR3 (each session 2 h at 0.1 mg/kg/infusion), before transferring to cocaine-SA on a PR schedule to test with retigabine.On the PR schedule (Figure 4A), three groups of rats self-administered cocaine at one of three different unit doses (0.06, 0.1 or 0.25 mg/kg/infusion) with 3 days of FR3 between PR sessions (method adapted from Allain et al. 31 ).See Data S1.

| Sucrose-SA
Naïve, ad libitum fed rats (n = 12) were trained on an FR1 sucrose-SA paradigm (20 s timeout post infusion) for 3 h per session (Figure 2A), one session per day, 6 days per week as previously described. 9See Data S1.

| Western blot
Using our published procedures, 9 we conducted a Western blot analysis to assess the expression of Kv7 channel isoforms in the NAc region (core and shell) and VTA of rats that underwent either cocaine (n = 4; Group 1) or sucrose (n = 4; Group 2) SA (see Figure 2).Membranes were blocked at room temperature for 1 h in Tris-buffered saline with 5% Bovine Serum Albumin (BSA) and 0.1% Tween20 then probed overnight with primary antibodies.See Data S1.These behavioural results identified a dose of retigabine sufficient to reduce noncontingent reward behaviour.

| Training to self-administer cocaine or sucrose
To determine whether retigabine would also reduce the operant responding for a drug relative to a natural reward, we trained separate groups of male rats (Figure 2A) to respond for cocaine (0.5 mg/kg/ infusion; Figure 2B) or sucrose pellets (Figure 2C) on an FR1 schedule of reinforcement for at least 10 consecutive sessions.The rats readily learned to distinguish between active and inactive cocaine levers To determine whether the cocaine experiences altered the protein expression of K v 7 channel subunits relative to sucrose, we sacrificed a subset of Group 1A sucrose-SA and cocaine-SA rats at 24 h after the final SA session for a Western blot analysis (Figure 2D).We evaluated the main subunits of the neuronal K v 7 family in the NAc and VTA tissue homogenates.In the NAc of cocaine-SA rats, the expression levels of K v 7.5 were reduced (Figure 2E; t 6 = 4.277, p = 0.0052), whereas the K v 7.2 (t 6 = 0.4209, p = 0.6885) and K v 7.3 (t 6 = 0.3348, p = 0.7491) were not.In contrast, in the VTA region, the F I G U R E 3 Retigabine reduces cocaine-but not sucrose-self-administration (SA).(A) Schematic depicting retigabine testing on a fixed-ratio 1 (FR1).Each group underwent 3 cycles (4 sessions per cycle) of testing with pretreatments with saline (1 mL/kg; Sessions 1, 2 and 4) or increasing doses of retigabine (2, 5, 7 mg/kg, i.p.; Session 3) in a repeated-measure design administered 15 min before SA.(B, C, D) Summary data showing active lever responding in consecutive sessions (expressed as a percent of baseline) during an FR1 reinforcement schedule for cocaine at unit doses of (B) 0.5 mg (C) 0.1 mg or (D) sucrose (45 mg).Retigabine altered the active-lever responding for (B, C) cocaine at both unit doses, but not for (D) sucrose.Grey bars indicate retigabine pretreatment (R) at doses denoted by the numbers in parentheses (2, 5, 7 mg/kg, i.p.).(E) Summary of the total reward consumed after pretreatment with saline or different doses of retigabine (▼2, •5 or 7 mg/kg).Significance compared with saline at the cocaine unit doses of 0.5 mg (7 mg/kg, **p = 0.0133) and 0.1 mg (2 mg/kg, **p = 0.0027; 5 and 7 mg/kg, ****p < 0.0001) using a Šídák's multiple comparison test.K v 7.2 (t 6 = 0.0074, p = 0.9943), the K v 7.3, t 6 = 0.4033, p = 0.7007) and K v 7.5 (t 6 = 0.7127, p = 0.5028) were similar between the drug and nondrug reward groups (Figure 2E).Based on these results, we next determined whether the retigabine activation of inhibitory K v 7 channels would reduce the operant responding for cocaine or sucrose.
To understand if the amount of reward consumed also decreased, we adjusted the change in the active lever responses for the total amount of reinforcer consumed in mg/kg and then expressed the data as a percentage of the baseline response to the preceding saline pretreatment (Figure 3E).A two-way RM model with factors of pretreatment (saline or retigabine) and dose of retigabine revealed for the highest dose of cocaine (Figure 3E, left) a significant main effect of retigabine treatment (F 1,90 = 5.795, p = 0.0181) and dose (F 2,90 = 11.40,p < 0.0001), with an interaction between treatment and dose (F 2,90 = 11.40,p < 0.0001).The lower dose of cocaine (Figure 3E, middle) showed a similarly significant effect of retigabine (F 1,10 = 71.71,p < 0.0001) and dose (F 2,20 = 6.295, p = 0.0076), with an interaction between treatment and dose (F 2,10 = 6.295, p = 0.0170).However, sucrose animals (Figure 3E, right) showed no such response to retigabine (treatment: F 1,11 = 0.2371, p = 0.6359; dose: F 2,22 = 1.287, p = 0.2962; interaction: F 2,22 = 1.287, p = 0.2962).These results indicated that the retigabine responses differed between the cocaine-and sucrose-SA groups.
With the higher dose of cocaine (Figure 3E, left; Group 1A: 0.5 mg/kg/infusion), responding on the active lever increased after 2 mg/kg retigabine and decreased after retigabine at 5 and 7 mg/kg.Compared with saline pretreatment, only the reduction in cocaine consumption with the 7 mg/kg dose of retigabine differed significantly ( Šídák's multiple comparison test, t 180 = 3.379, p = 0.0133).
The changes in cocaine consumption with the 2 and 5 mg/kg dose of retigabine did not differ significantly compared with saline ( Šídák's multiple comparison test, 2 mg/kg t 180 = 2.564, p = 0.1551; 5 mg/kg t 180 = 2.815, p = 0.0783).On the contrary, with the lower dose of cocaine (Figure 3E, middle; Group 1B: 0.1 mg/kg/infusion), cocaine consumption decreased significantly relative to the saline baseline in all rats (2 mg/kg, t 20 = 4.589, p = 0.0027; 5 mg/kg, t 20 = 6.541, p < 0.0001; 7 mg/kg, t 20 = 7.789, p < 0.0001).The retigabine reduction was $3.5-fold greater with 7 mg/kg than 2 mg/kg retigabine Because the retigabine action did not generalize across reinforcers, we inspected the cumulative records of individual animals within the various reinforcer SA sessions (Figure 3B-D) and made some general observations.First, we noted that the reductions related to retigabine in the distribution of the inter-event intervals (Figure S1) for cocaine appeared dose-dependent.That is, in general for the cocaine sessions, the total number of infusions decreased as the dose of retigabine increased.However, retigabine failed to affect the distribution of inter-event intervals at either concentration of cocaine.Second, we observed that these changes were not evident in sucrose-SA rats.As an additional step, we evaluated responding on the inactive lever during pretreatment with saline for the three increasing doses of retigabine.A 2W RM ANOVA did not indicate significant differences related to retigabine in inactive lever responses within cocaine (Groups 1A and 1B) or sucrose (Group 2) rats (Figure S2).Taken together, these results indicate that the pharmacological enhancement of K v 7 channels with retigabine reduced cocaine-SA, but the extent to which this occurred depended on both the dose of cocaine and retigabine.Lastly, the effects of retigabine did not generalize to sucrose reward.

| Retigabine testing in a progressive-ratio task
The PR schedule provides an index of reinforcement efficacy. 32As a final test, we used this approach to determine whether enhancing the activity of K v 7 channels altered the motivation to earn a cocaine reward.Using Group 1C rats, we compared PR responses (Figure 4A) after a pretreatment with saline (1 mL/kg, i.p.) versus retigabine (5 mg/kg, i.p.) in a repeated measure design.We used separate groups of animals to test the different doses of cocaine (0.06, 0.1 or 0.25 mg/kg/infusion).All rats reached the breakpoint prior to the 3-h time limit.During PR tests, we found that responding for cocaine was dose-dependent and that rats responded less for cocaine at lower doses (Figure 4B; 2W RM mixed-effects model: F 2,29 = 35.79,p < 0.0001).There was also a main effect of treatment (Figure 4A), indicating that when averaged between cocaine doses, rats exhibited lower infusion rates when treated with retigabine than when treated with vehicle (F 1,25 = 29.01,p < 0.0001).The difference between retigabine and vehicle was robust at a low dose of cocaine, but only modest at the highest dose of cocaine tested.There was a significant interaction (Figure 4B) of the dose of cocaine with retigabine (F 2,25 = 3.627, p = 0.0414).A post-hoc Šídák's multiple comparison tests indicated that retigabine reduced infusions at the two lowest unit doses of cocaine (Figure 4B; 0.06 mg unit dose: t 25 = 4.696, p = 0.0002; 0.1 mg unit dose: t 25 = 3.857, p = 0.0021; 0.25 mg unit dose: t 25 = 0.9256, p = 0.7421).Across the groups, the cocaine breakpoints increased as a function of the cocaine dose (main effect, F 2,29 = 12.71, p < 0.0001; Figure 4C).However, the dose-response curve for cocaine was significantly decreased in response to retigabine (retigabine F 1,24 = 10.63,p < 0.0033; dose Â retigabine F 2,24 = 0.7867, p = 0.4667; Figure 4C).There were no main effects for the responses of the inactive lever.In summary, retigabine significantly reduced cocaine intake over time and reduced incentive motivation for the drug.In contrast, at the doses tested, retigabine produced little measurable change in sucrose-SA.

| DISCUSSION
We identified K v 7 channels as a regulator for the targeted control of chronic cocaine-SA.On a fixed-ratio reinforcement schedule, systemic treatments of retigabine dose-dependently reduced leverpressing for cocaine but not the 'natural' reward, sucrose.These observations led us to pharmacologically evaluate K v 7 channel activation during a PR schedule of reinforcement, determining how retigabine shaped motivational responses at different doses of cocaine.Indeed, pretreatments with retigabine obstructed the motivational effects of cocaine at low to moderate doses.Therefore, we posit that, in combination with counselling and behavioural therapies, targeting K v 7 channels may be a useful strategy for reducing the early stages of drug use and complementing other treatments aimed at recovery during prolonged withdrawal. 33The present findings extend our previous research demonstrating that direct activation of cortical K v 7 channels with retigabine reduced relapse-like behaviours 9 and align with recent accumbal work examining experimenter-delivered drug behaviours. 11

| Retigabine reduces cocaine CPP
To confirm an in vivo dose that would be effective in operant experiments, our research demonstrated a systemic dose of retigabine that blocked the acquisition of cocaine CPP.The reduction resembled that previously reported with flupirtine, 34 a less selective structural precursor of retigabine that activates K v 7.2/7.3and other channels. 35e behavioural reduction with retigabine may reflect decreased DA transmission, as similar doses of retigabine reportedly decreased the enhancement of striatal and cortical DA levels associated with noncontingent administration of cocaine, 16 which appear necessary for the acquisition of CPP. 36

| Retigabine actions in cocaine-or sucrose-SA behaviour
We found that at lower doses of cocaine, K v 7 channel activation reduced operant responses that had been learned and repeatedly reinforced in an FR paradigm.Although previous studies have not investigated this using operant behaviour, retigabine reduced ethanol consumption in a two-bottle choice study 37 and alcohol phenotypes associated with K v 7 channels. 38[45] In addition to the examination of fixed-ratio responses, we confirmed the action of retigabine by measuring the motivational changes to obtain cocaine in a PR reinforcement schedule. 46After confirming a previous PR study showing that 0.06-0.25 unit doses supported cocaine-SA, 31 we showed that retigabine was more effective at lowering PR responses in lower doses of cocaine.9][50] PR responses for cocaine follow the same pattern. 32,51like cocaine, retigabine did not reduce sucrose-SA on a fixedratio schedule of reinforcement, which was relevant given that both rewards are associated with cue-induced seeking 52 and DA neuron activation. 53However, although DA release is time-locked to approach behaviour for both cocaine and sucrose, DA levels quickly normalize during SA of sucrose, but not cocaine. 54,55Furthermore, unlike sucrose-SA, rats with a history of chronic cocaine-SA do not show the normal decrease in the firing of VTA DA neurons that occurs with learning. 56Furthermore, while cocaine-SA produces persistent long-term potentiation (LTP) in VTA DA neurons, sucrose-SA does not. 57Although retigabine-LTP studies have not been reported in VTA DA neurons, in vivo administration of retigabine or flupirtine prevents hippocampal LTP and this effect was blocked by the selective K v 7 channel antagonist XE991. 58Furthermore, our previous work and others showed that direct cortical application of retigabine blocked the seeking for cocaine but not sucrose, the former of which requires VTA DA signalling in the prelimbic cortex. 59,60

| Expression of K v 7 channel subtype after rewards
Our results confirmed earlier studies showing the expression of K v 7 channel subunits in NAc and VTA. 27,61,62Unlike other reports linking decreases in brain K v 7.2/K v 7.3 protein levels with alcohol withdrawal 63 or a depressive phenotype, 13,24,27 we found no measurable differences between the drug and non-drug reward groups in the expression of K v 7.2/K v 7.3.7][68] Chronic upregulation of cAMP-PKA signalling is associated with reward tolerance and dependence that drive SA. 65 However, over time, chronic overactivation of cAMP-PKA signalling could produce homeostatic decreases in K v 7.5 protein expression.[71]

| CONCLUSIONS
Our behavioural data align with published behavioural and electrophysiology work causally linking K v 7 channel-mediated inhibition to reductions in DA neuron activity. 10Doses of retigabine similar to those used in our study reduced the frequency of VTA action potential firing and reduced psychostimulant-induced increases in DA release from the terminals. 15,20In hippocampal and VTA DA cells, retigabine more potently suppressed burst firing activity than basal neuron firing. 20,72Therefore, a reasonable scenario is that retigabine activation of K v 7 channels attenuated excessive dopaminergic neurotransmission in the mesolimbic system, to reduce cocaine-SA.
Although our data provide a more inclusive evaluation of retigabine and SA is a well-validated model with predictive value for medications that effectively treat cocaine dependence, the utility of retigabine for human substance abuse disorder is unclear.Whereas preclinical studies often use acute medication pretreatments to evaluate SA, most human studies instead assess cocaine craving or 'high'. 73 controlled clinical trials, cocaine-SA appears difficult to diminish and may differ at different stages of the addiction cycle.Encouraging examples that decrease cocaine use include modafinil, baclofen and buprenorphine, although the latter two are limited by the issues of dose or enhancement of cocaine intoxication. 739][80] More selective versions of this drug may be promising for the treatment of hyperexcitability diseases. 81 All values are expressed as means ± SEM.The normality of the data was assessed and found to be normally distributed according to a D'Agostino and Pearson omnibus normality test.Differences between groups were assessed using t-tests, one-way or two-way repeated measures (RM) mixed-effects model.When significant main effects were obtained, appropriate post-hoc comparisons between groups were made using a Fisher's LSD, Šídák's or Tukey multiple F I G U R E 1 Retigabine reduces the acquisition of cocaine conditioned place preference (CPP).(A) CPP testing included a pretest and 5 days of twice daily intraperitoneal injections of cocaine (black box; 10 mg/kg) and saline (white box; 1 mL/kg) in alternating counterbalanced fashion.At 15 min before cocaine, rats received intraperitoneal pretreatments of either vehicle (1 mL/kg; n = 11) or retigabine (5 mg/kg; n = 11).On Day 7, rats were tested for side preference.(B) CPP score was calculated as the difference in the time spent in individual chambers during pre-test and post-test.A 2W ANOVA indicated an interaction of conditioning with cocaine Â retigabine (F (1, 40) = 4.551, p = 0.0391).Rats pretreated with vehicle showed a preference for the cocaine-paired chamber (t (40) = 3.530; **p = 0.0011) compared with pre-test.Rats pretreated with retigabine showed reduced preference for the cocaine-paired chamber compared with vehicle-treated controls (t (40) = 2.567 *p = 0.0141).For these and all other figures, the error bars indicate the mean ± SEM. comparisons test.Significance was set at α = 0.05.All analyses were performed on Prism version 9.4.0 (GraphPad, La Jolla, CA).
but its functional impact on cocaine-SA remains unknown.To evaluate this, we used a cocaine CPP (Figure1A) assay to first confirm a retigabine dose that effectively reduced the acquisition of a noncontingent reward behaviour.For 5 days, we pretreated rats with retigabine (5 mg/kg, i.p.) or vehicle 15 min before daily counterbalanced treatments of cocaine or saline.A mixed effects ANOVA showed a significant interaction of conditioning Â retigabine (F (1, 40) = 4.551, p = 0.0391).During a post-test, rats pretreated with vehicle showed a preference for the cocaine-paired chamber (t (40) = 3.530; **p = 0.0011) compared with the pre-test.When animals were pretreated with retigabine, rats spent significantly less time in the cocaine-paired chamber compared with vehicle-treated controls following cocaine conditioning (Figure1; t (40) = 2.567; *p = 0.0141).
Taken together, our work emphasizes the potential value of future clinical trials with retigabine and further identification of its mechanisms relevant to drug SA.AUTHOR CONTRIBUTIONS Esteban S. Urena: Conceptualization; methodology; data collection; writing-original draft; writing-review and editing.Cody C. Diezel: Conceptualization; methodology; data collection; writing-review and editing.Mauricio Serna: Data curation; data collection; writingoriginal draft; writing-review and editing.Grace Hala'ufla: Data collection.Kara R. Barber: Data collection; data curation; conceptualization; methodology; writing-original draft.Lisa Majuta: Data collection; methodology; supervision; writing-review and editing.Todd W. Vanderah: Writing-reviewing and editing.Arthur C. Riegel: Conceptualization; writing-review and editing; supervision.